(17d) Enzyme-Encapsulating Polymeric Nanoparticles for Treating Glutamate Excitotoxicity

Introduction: Despite the neurological advancements of the past century, treating many neurological diseases remains elusive and costs $750 billion annually in the United States alone. Most neurological diseases, including stroke, traumatic brain injury, and Alzheimer’s, exhibit glutamate excitotoxicity (GluEx). Glutamate is a major neurotransmitter used for neuronal communication, but leads to neuronal death when in excess by over-activating glutamate receptors to eventually induce apoptosis. Following neuronal death, neurons release more glutamate, causing further neighboring neuronal death. Stopping the propagation of the GluEx cycle is crucial for preventing additional damage and promoting recovery. However, successful treatment of GluEx requires a therapeutic that not only biochemically functions appropriately, but also effectively reaches the target site past obstacles that adversely influence therapeutic efficacy, including systemic clearance and crossing the highly restrictive blood-brain barrier (BBB). Even after overcoming these two barriers, a therapeutic must be capable of diffusing through the brain parenchyma to reach diseased target sites. Because of the complexity of treating neurological injury, testing therapeutics on simplified yet representative models with intentionally eliminated barriers provides a systematic approach towards evaluating drug efficacy. By evaluating nanoparticle behavior and drug delivery efficacy in a model that captures the in vivo brain microenvironment, we can develop effective therapeutic NPs for site specific delivery to only regions of injury in the diseased brain.

Materials and Methods: We formulated enzyme-encapsulating polymeric nanoparticles (EE-NPs) using the double emulsion method. Catalase was selected as our initial enzyme due to its promising antioxidant properties of decomposing hydrogen peroxide (H₂O₂) into water and oxygen to combat GluEx. We encapsulated catalase within poly(lactic-co-glycolic acid)-block-poly(ethylene glycol) (PLGA-PEG) co-polymer. We also developed an organotypic whole hemisphere brain slice model of GluEx. In this model, we incubated slices with the known glutamate receptor toxin N-methyl-D-aspartate (NMDA) to initiate neuronal death to imitate the GluEx environment. We quantified lactate dehydrogenase (LDH) release, propidium iodide (PI) staining, and Fluoro-Jade C staining, as well as H₂O₂ and glutamate concentrations. These measurements enabled us to characterize cell cytotoxicity and potential biomarker concentrations. We then combined these two platforms to test the efficacy of the EE-NP in decreasing cell death and toxic biomarker concentrations in our slice model. We utilized empty PLGA-PEG nanoparticles and free catalase as controls.

Results and Discussion: We formulated nanoparticles with a catalase/PLGA core and a dense PEG coating to achieve a hydrophilic, bio-inert surface. The EE-NP exhibited rapid diffusion in brain tissue due to its non-binding surface and sub-100 nm size, and controlled linear release of catalase over 48-72 h. With the methods previously stated, we obtained a characteristic profile of cell viability and biomarker environment for GluEx-induced brain slices. After application of EE-NPs to the NMDA treated brain slices, we observed decreased cell death and lower glutamate and H₂O₂ concentrations. This work establishes a therapeutic/ex vivo brain slice model that semi-quantitatively assesses extent of cell death and biomarker concentrations representative of the in vivo environment. Our work provides evidence of EE-NP efficacy, supporting catalase EE-NPs as a potential therapeutic for glutamate excitotoxicity.

Conclusions: Treating neurological diseases has proved incredibly difficult, largely due to ineffective drug delivery. Before we can develop effective therapeutics ready for market, greater understanding of a drugs ability to circumnavigate barriers in the brain is required. The ex vivo model permits evaluation of nanoparticle diffusion to target sites through brain extracellular space, and subsequent controlled enzyme release, and catalase therapeutic efficacy. Beyond promoting catalase EE-NPs as a potential GluEx therapeutic, our findings support the systematic approach of developing ex vivo models that selectively capture barriers to neurological drug delivery, and fine-tuning the design of therapeutics to overcome each barrier. This slice model retains the complexity of the brain parenchyma while bypassing systemic and BBB obstacles. By gradually reintroducing complexity to the ex vivo model until it transitions to in vivo, we can isolate bottleneck barriers and overcome them one at a time to ultimately design a fully effective therapeutic for glutamate excitotoxicity.